Signal enhancement of surface plasmon-coupleddirectional emission by a conical mirror
Derek S. Smith, Yordan Kostov, and Govind Rao*Center for Advanced Sensor Technology and Department of Chemical and Biochemical Engineering, University of Maryland
Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250, USA
The use of fluorescence spectroscopy has steadily in-creased in chemical, biochemical, and medical re-search with applications including examiningvarious analyte concentrations in cell cultures andfermentations [1–6], DNA and RNA detection and se-quencing [7,8], and protein microarrays [9] and im-munoassays [10–12]. However, there is always ademand for increased sensitivity to lower the detec-tion limits of these assays. Numerous methods havebeen used to achieve this, including the various fluor-escence observables. Yet, the sensitivity is alwayslimited by background luminescence, the collectionefficiency of the detection system, and photodeter-ioration of the fluorophores used.Current efforts to generate greater sensitivity
have focused on the effects of metallic nanoparticlesin close proximity to fluorophores. These subwave-length particles have a twofold effect on the fluoro-phores, simultaneously providing improvedphotostability by decreasing the lifetime and in-creased sensitivity by enhancing the quantum yield
[13–20]. This is described in the literature as metal-enhanced fluorescence (MEF) with applications ran-ging from bioassays to pH sensing [21–25]. Despitethese enhancements, the MEFmethod is still limitedby background fluorescence and the collection effi-ciency of the system.
Another plasmonic phenomenon occurs in the pre-sence of subwavelength metallic films. According tothe Radiating Plasmon Model [26], surface plasmon-coupled emission (SPCE), or surface plasmon field-enhanced spectroscopy, is observed when excitedfluorophores induce surface plasmons in a nearbymetal film through a nonradiative energy transfer[27–35]. The plasmons then radiate the energy withthe same spectral distribution as the fluorophores.The resulting far-field emission is highly directionalat a specific angle based on the optical properties ofthe system. This is due to physical constraints thatonly allow electromagnetic waves of a particular or-ientation and polarization to excite surfaceplasmons.
The underlying difference between typical fluores-cence observation and SPCE is the directionality ofthe emission. One of the limiting constraints on fluor-escence spectroscopy is the isotropic nature of the
radiation. This generates low collection efficiency un-der normal observation conditions (approximatelyonly 1% of the total emission is collected) [36]. Onthe other hand, the polarized, directional SPCE en-hances the collection efficiency with conventional ob-servation methods, leading to the measurement offluorescence from monolayers [37,38] and single mo-lecules [39,40]. Typical enhancements over the freespace, isotropic emission range from 10 to 20 fold;however, this is only due to observation of a smallfraction of the SPCE. Current observation techni-ques do not collect the entire SPCE ring (as observedwhen the emission is coupled through a hemispheri-cal prism [28,37]) with a single detector. Conse-quently, only approximately 1=180th of the signal(2 arc deg) is collected. If it were possible to collectmost or all of the SPCE, it is estimated that signalenhancements of 103–104 could be achieved [28].In this paper, we describe a simple optical method
to focus the directional SPCE into a single point, al-lowing for collection of most or all of the coupledemission. By placing a conical mirror around a hemi-spherical coupling prism, we were able to direct theSPCE ring from approximately a 50nm poly(vinyl al-cohol) (PVA) layer doped with ruthenium into a sin-gle point. This resulted in nearly a 500 foldenhancement over the free space signal. The signifi-cantly increased intensity led to a nearly 35 fold in-crease in the signal-to-noise ratio as well. Thesensitivity gained using standard optical piecesdue to the focusing of the SPCE ring into a singlepoint coupled with LED excitation [41] would allowfor the development of low cost, hand-held fluores-cence devices with a wide range of applications.
2. Experimental Methods
A. Sample Preparation
A 50nm thick layer of silver, followed by a 5nm thicklayer of SiO2, vapor-deposited on a glass microscopeslide (plain, Corning, Corning, New York) was ob-tained from EMF Corporation (Ithaca, New York).The SiO2 layer serves two purposes: It protects thesilver layer from oxidation and creates a spacer fromthe metal film to prevent quenching. An approxi-mately 1 μm solution of tris-(bathophenanthroline)ruthenium(II) chloride (GFS Chemicals, Columbus,Ohio) in 1% PVA (Sigma-Aldrich, St. Louis, Missouri)was spin-coated on the surface of the slide to deposita 50nm fluorescent layer.The conical mirror was machined from stainless
steel at a local machine shop. The surface roughnessof the cone was on the order of 20 μm. The angle of thecone was designed to be 70° with an inner diameterto accommodate a 1 in: (2:54 cm) hemispherical lens.The mirror was then hand polished in the laboratoryusing diamond-lapping pastes from McMaster-Carr(Aurora, Ohio). The final polishing compound usedwas Masterpolish Final Polishing Suspension fromBuehler (Lake Bluff, Illinois).
B. Fluorescence Measurements
The sample slide was attached to a BK7 prism withglycerol as the indexmatching fluid (schematic shownin Fig. 1). For typical observation of SPCE, the spin-coated slide was attached to a hemicylindrical prismandplaced on a rotational stage, allowing observationat all angles relative to the vertical axis. For SPCEobservation using the new configuration, the conicalmirror was placed around a hemispherical prism,which was then mounted on the rotational stage.The mirror was designed to focus the SPCE ring intoa single point as shown in Fig. 2. Additionally, a black,circular rubber coating was painted onto the center ofthe prism to block any direct laser light interference.All the measurements presented were performed inthe reverse Kretschmann (rKR) configuration, wherethe sample fluorophore is excited directly (shown inFig. 1). It is important to note that surface plasmonsare not excited by this method of incident illumina-tion. Rather, the excited fluorophores induce surfaceplasmons, which then radiate the coupled energythrough the prism. There is another mode of excita-tion, termed the Kretschmann (KR) configuration,where illumination occurs from the prism side ofthe sample. In this case, the light is incident at thesurface plasmon angle for the system creating surfaceplasmons. Here, the evanescent field of the plasmonscauses the excitation of the fluorophores. The result-ing far-field emission has the same spectral distribu-tion as the free space fluorescence.
Excitation was achieved with a 405nm, 3mWsemiconductor laser diode (TE cooled module, Photo-nics Products) passed through an 8mmdiameter slit.
Fig. 1. (Color online) Schematic of the sample configuration. Re-verse Kretschmann (rKR) excitation from a 405nm laser was usedto directly excite ruthenium molecules in the PVA layer. The re-sulting SPCE exited the hemispherical prism at 56:5° in the shapeof a ring and was reflected by the conical mirror into a point thatwas observed with a liquid light guide fiber optic.
The emission was observed through a 550nm LWPfilter placed at the end of a 3mm diameter liquidlight guide (Oriel UV-Vis, Newport, Stratford, Con-necticut) that was positioned either on the sampleside for free space measurements, at the SPCE angle,or at the focused point of the SPCE ring after reflec-tion. The output of the light guide was sent directly toa monochromator attached to a modified ISS K2 fluo-rometer (Champaign, Illinois) with single-photoncounting capabilities. For angularity measurements,a 20 μm vertical slit was placed on the fiber to in-crease angular resolution.
3. Results and Discussion
A. Surface Plasmon-Coupled Directional EmissionCharacteristics
Prior to testing the proposed method for reflectingthe SPCE ring into a single point, the sample wasanalyzed to verify its SPCE characteristics. First,the directionality of the emission was observed.Using the equations from surface plasmon resonancetheory, the reflectance profile for the radiated lightfrom SPCE can be calculated. The reflectance mini-mum corresponds to the angle at which the radiationcan be expected to occur for the system. These equa-tions can be found in the literature [42,43]. Reflec-tance calculations can also be performed usingTFCalc 3.5 software (Software Spectra, Portland,Oregon). Figure 3 shows the calculated reflectivitycurve generated from the software (bottom) for thesystem shown in Fig. 1 as well as the observed angu-lar profile of the SPCE (top). Theory predicted an an-
gle of 56:55° for the system, while observationindicated an angle of 56:5°. This fractional displace-ment of the SPCE angle compared to the SPR pre-dicted angle has been previously reported in theliterature [27,44,45]. Note that the emission ismostly confined within approximately 2–4 arc degas expected for SPCE.
Analysis of the emission spectrum was also per-formed to further characterize and verify the SPCEfrom the sample. First, the fiber was positioned tomeasure the free space emission from the sampleside. Following this, the fiber was rotated to theSPCE angle on the prism side of the sample to mea-sure the SPCE spectrum. The resulting spectra aredisplayed in Fig. 4. Note the noise associated withboth the free space and SPCE angle measurementswithout the focusing ring. Comparing the peak inten-sity, the directionality of the SPCE led to a signal in-crease of about sevenfold over the free spacemeasurement. This enhancement falls within the ty-pical range of laser excited SPCE, where reportshave shown 3 to 27 fold enhancements [27,29,38].
Fig. 2. (Color online) Images of the ruthenium SPCE taken witha screen positioned at different distances from the focal point.(a) SPCE ring as viewed at themirror/emission reflection. (b) SPCEfocused point as projected onto a screen of OD∼ 2. (c) SPCE ring asprojected onto a screen of OD∼ 2. (d) Copy of Fig. 2(c) with theintensity enhanced for clarity.
Fig. 3. (Color online) (Top) Observed angular distribution of theSPCE from a 50nm PVA layer doped with ruthenium. (Bottom)Calculated reflectance profile of the system shown in Fig. 1 forthe emission wavelength of 600nm.
Also, the plasmon-coupling did not distort the spec-trum, which would be important in any type of sen-sing application. Figure 4 also shows that, despitethe increased collection efficiency of SPCE comparedto the free space, there is still visible noise in the ob-served signal from SPCE without the focusing ring.The polarization of the emission was also mea-
sured as a final verification of SPCE. Spectra weretaken (not shown) at the SPCE angle with a polarizerin front of the observation fiber. The polarizer wasoriented to allow only p-polarized or s-polarized lightto pass in two different measurements. It is expectedthat the SPCE should be highly p-polarized due tothe wavevector matching requirements of SPR theo-ry that allow only p-polarized incident light to createplasmons. Indeed this was the case, as the emissionwas 98% p-polarized, corresponding to IVH=IVV ¼ 96.This interesting property could be used to furtherlimit the amount of unwanted light that could inter-fere with measurements.
B. Mirror Collected Surface Plasmon-Coupled DirectionalEmission
After verifying that the emission from the sample ishighly directional, p-polarized SPCE, the observa-tion setup was modified by placing a conical mirrormade of polished stainless steel around a hemisphe-rical prism as shown in Fig. 1. Since the emissionpropagates in the form of a ring, as shown in Fig. 2(a), it was postulated that, by using a ring-shapedmirror, the collection efficiency could be greatly en-hanced, leading to the realization of the 103–104 en-hancement factors that were predicted for SPCE[28]. The conical shape of the mirror was chosen inorder to reflect the light into a point at angles thatwere consistent with the acceptance angles of the li-quid light guide fiber optics used. Also important isthe fact that any axial displacement from the centerof the conical mirror does not result in significant dis-
tortion of the focused point. Rather, it results in a si-milar planar displacement from the expected focalpoint. This would not be the case if, for example, aparabolic mirror had been chosen. In our case, withthe mirror angled at 70° and the SPCE emitted at56:5°, the light reaches the fiber optic at an angleof 16:5°, well within the 36° acceptance angle ofthe fiber used.
The results of the conical mirror collection methodcan be seen in Fig. 2(b), which shows a photo of thefocused point projected onto a screen. For compari-son, the screen was moved toward the mirror creat-ing the projection of the SPCE ring shown in Fig. 2(c).Due to the low intensity observed, the image was en-hanced for clarity as shown in Fig. 2(d). Notice thedifference in intensity between the focused pointand the ring when projected onto the screen. It isclear that the mirror is acting like a lens for theSPCE. The increased intensity visually observed atthe focal point indicates the configuration is workingas desired.
In order to determine the effectiveness of the mir-ror, the fiber optic was placed at the maximum inten-sity position of the focused point, and the spectrumwas measured as shown in Fig. 4. Note the differ-ences in the left and right y-axis scales. The reflect-ion of the SPCE ring into a single point (Fig. 2(b))resulted in close to a 73 fold enhancement of theSPCE signal collected under normal observationconditions. In other words, the observation methodresulted in nearly a 500 fold total signal enhance-ment over the free space signal. However, this largeenhancement is actually lower than expected. Thiscan be attributed to multiple causes. First, the reflec-tance of stainless steel is approximately only50–60%. We are currently designing future proto-types that will be made of metals with higher reflec-tance such as aluminum. Second, the polishingwas performed in house and was difficult due tothe odd shape. Consequently, future prototypes willbe professionally polished to obtain the maximumreflectance possible. With these modifications, thistechnique should allow for a 1000 fold increase ormore over the free space intensity.
The signal-to-noise ratio was also calculated fromthe measured spectra. The spectra shown in Fig. 3were not smoothed in order to show the amount ofnoise present in the free space and SPCE measure-ments. All spectra were taken at the same distanceaway from the sample. The overall result was an in-crease in the signal-to-noise ratio of nearly 35 foldwhen comparing the free space signal to that ofthe focused SPCE point. This increase is largerthan expected and can possibly be attributed tothe thermal stability of the laser excitation and gen-eral instrumentation noise. While this increase isextremely beneficial, we are currently investigatingthe source of the noise discrepancy.
Fig. 4. (Color online) Ruthenium emission spectra due to rKR ex-citation as observed from the free space, the SPCE angle, and thefocused SPCE point. No smoothing was performed for any curves.The use of the conical mirror resulted in nearly a 500 fold signalenhancement, leading to almost a 35 fold increase in the signal-to-noise ratio as compared to the free space signal.
The optical setup presented here allows for enhance-ment of the surface plasmon-coupled emission offluorophores positioned in close proximity of thin me-tal films while generating large increases in the sig-nal-to-noise ratio. The intensity and signal-to-noiseratio gains achieved by further enhancing the collec-tion efficiency of the directional fluorescence asso-ciated with surface plasmons allow for increasedsensitivity with far less complex instrumentationas currently needed. Coupling this setup with thepossibility of LED excitation [41] could lead to thedevelopment of low cost, high sensitivity portable de-vices with rapid response times. One of the manypossible applications is the development of ultrafastSPCE-based sensing schemes [38].
This work was made possible by funding from theNational Science Foundation, Division of Bioengi-neering and Environmental Systems, grant award00517785.
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